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The CHROMacademy Essential Guide Webcast:
HPLC Detectors – What, Where, When, and How.

Thursday, January 23rd 2014, 16:00 GMT.

The selection of a detector for HPLC is based on the chemical nature of the analytes and any potential interferents that may be present, the limit of detection required, and often the availability and cost of the detector. This month’s Essential Guide will focus on the operating principles of the most common HPLC detectors. The parameters that should be optimized for each detector type will be examined to give the optimum performance from the chosen detector. The advantages, disadvantages, and application areas for each detector type will also be discussed. This in depth review of HPLC detectors will result in a better understanding of the operation, optimization, and detector choice for every application.

  • What detectors are available for HPLC applications
  • When and why would you choose one detector over another
  • What are the advantages and disadvantages of each detector type
  • Which detector parameters require optimization and what is their impact?
  • Is there an ideal detector and if not what would it look like?
 

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Who Should Attend:

  • Anyone working with HPLC who would like to better understand the common detectors in use
  • Anyone who wants to better understand the factors that affect HPLC detection methods
  • Anyone involved in developing HPLC methods
   


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The CHROMacademy Essential Guide Tutorial
HPLC Detectors – What, Where, When, and How - Available Late January 2014

The selection of a detector for HPLC is based on the chemical nature of the analytes and any potential interferents that may be present, the limit of detection required, and often the availability and cost of the detector. This month’s Essential Guide will focus on the operating principles of the most common HPLC detectors. The parameters that should be optimized for each detector type will be examined to give the optimum performance from the chosen detector. The advantages, disadvantages, and application areas for each detector type will also be discussed. This in depth review of HPLC detectors will result in a better understanding of the operation, optimization, and detector choice for every application.

  HPLC Detectors
  • Understand the operating principles and limitations of common HPLC detectors
  • How to optimize detector settings to maximize analytical response
  • Identify the correct detector for a specific application
  • Understand the importance of using the correct detector settings for your analysis
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Selectivity

Non-selective detectors react to the bulk property of the solution passing into the detector (i.e. refractive index detector).  When a compound elutes from the column there is a change in the bulk property which can be measured and recorded.
Selective detectors do not react to the bulk solution passing through the detector; instead a response due to a specific property of the solute molecule is measured (i.e. a chromophoric moiety in UV absorbance).

Sensitivity

The smallest detectable signal is estimated to be equivalent to three times the height of the average baseline noise (Figure 1), giving a signal to noise ratio of 3:1 for the ‘Limit of Detection’ (LOD) of the detector.  If the amount of analyte injected is less than this the signal will be indistinguishable from noise.
For quantitative analysis a signal to noise ratio of 10:1 is recommended for the ‘Limit of Quantification’ (LOQ).

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Figure 1: Sensitivity and signal to noise ratio.
 
 

Limit of Detection/Quantification (LOD/LOQ)

Important terms associated with HPLC detectors are outlined in the interactive diagram below (Figure 2).

 

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Figure 2: Detector calibration plot.
 
 

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The Flow Cell

As the mobile phase leaves the analytical column it enters and fills the flow cell (Figure 3).  Light from the UV (Deuterium) or Visible (Tungsten) lamp shines through the flow cell and its contents.  The intensity of the emergent light from the flow cell is measured using photodiodes, which produce an electrical signal when exposed to light.  The greater the intensity of the light that reaches the photodiodes the larger the resultant signal.
A UV-visible detector actually measures the transmittance intensity (i.e. the light that is not absorbed by the sample) and NOT absorbance. Analytes which contain a UV chromophore(s) will cause large absorbance differences when they elute into the flow cell and the light exiting the flow cell will reduce markedly, generating a large response.  The electrical signal is constantly measured to produce a plot of Absorbance vs. Time.

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Figure 3: UV-visible detector flow cell.
 
 

Quantitation

Quantitation using a UV-visible detector is possible as absorbance is proportional to analyte concentration within the limitations of the Beer-Lambert Law
(Equation 1).

Where:
A = absorbance
ε = molar absorptivity (Lmol-1cm-1)
b = path length (cm)
c = concentration (molL-1)

 

When a plot of peak height or area versus concentration is made, the Beer-Lambert law dictates that as long as the determinations are carried out under identical conditions, the amount of an analyte within an unknown sample may be directly interpolated from this curve.  Determination of the concentration of an unknown sample is simplest in the linear region of the calibration curve.  However, linearity is not essential as long as the response is proportional, precise and demonstrates a good fit with an established mathematical transformation.  It is not uncommon for certain analytes, or even detectors, to produce quadratic, exponential or even sigmoidal curves.

For most HPLC analyses peak areas are used for quantitative calculations, although in most instances peak heights will give the same result.  Peak area is especially useful because HPLC peaks may be tailed.  Furthermore, if there are any changes in retention during a run, a specific analytes peak height may change fairly considerably, whereas, its peak area would not really change at all.  In these case peak heights may vary; however, peak area will remain constant resulting in more repeatable values.

Peak height calculations may be better for trace analysis when the peak height is very small or when peaks are poorly resolved.  The use of peak height will reduce the error sustained in small changes in peak start and end time variation.

The area of the chromatographic peak will change if there are changes in flow rate (Figure 4); therefore, it is essential that pumps are well maintained to ensure that flow rates are precise and stable.

Both peak height and area will be irreproducible if injection volume varies.

 
Figure 4: Variation of peak height and area with changes in flow rate. 
 
 

UV Chromophores

The visible and UV spectra of organic molecules result from transitions between electronic energy levels.  The wavelength of absorption is a measure of the separation of these energy levels.  Above 200 nm excitation of electrons from p-, d-, and π-orbitals, and especially, conjugated systems produce informative spectra. 
Electrons tightly bound in single C-C or C-H bonds absorb electromagnetic energies corresponding to wavelengths less than 180 nm which is below the useful operating range for a typical UV-visible detector. 

Analyte molecules containing only C-C or C-H bonds do not show high sensitivity in UV-visible detectors.  Heteroatoms such as sulfur, bromine, nitrogen, oxygen etc. have unpaired electrons which exhibit larger absorbances in the operating range of a UV-visible detector.  Electrons within unsaturated systems such as double or triple bonds are relatively easily excited by UV radiation giving rise to useful absorbance spectra.

Below approximately 200 nm it is possible for the solvent used in the mobile phase to interfere with the analyte absorbance measurement, therefore, this should be taken into account when selecting an analytical wavelength to monitor.

Absorbance wavelengths for some common functional groups and solvents are shown below (Figure 5).

 

Figure 5: Absorbance wavelengths for common functional groups and solvents.

 
 

Variable Wavelength UV-visible Detectors - Optical Path

Polychromatic light from a deuterium or tungsten lamp is focused onto the entrance slit of a monochromator using spherical and/or planar mirrors (Figure 6).  The monochromator selectively transmits a narrow band of light to the exit slit.  The wavelength of measurement is selected via the data system; this is achieved using a grating mounted on a turntable.  The grating is positioned to allow light of the correct wavelength, and only this wavelength, to pass to the second mirror and so on through the flow cell or the reference diode.  The magnitude of analyte absorbance is determined by measuring the intensity of light reaching the photodiode without the sample (reference) and comparing it with the intensity of light reaching the photodiode after passing through the sample.

 
 

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Figure 6: Variable wavelength UV-visible detector optical path.
 
 
Advantages
  • UV-visible detectors are sensitive and can detect analytes in the nanograms range (0.5-1.0 ng). 
  • As has been shown with the Beer-Lambert Law, UV-visible detectors can be used for quantitative analysis as analyte concentration is proportional to absorbance. 
  • UV-visible detectors respond to a physicochemical property of the analyte and not the bulk elution media, therefore, they are suitable for use with gradient analyses. 
  • A UV-visible detector will respond to many different types of analytes as long as there is a UV chromophore present.
Disadvantages
  • Absorption is dependent on solution conditions.  Solvent effects will lower the observed wavelength of an analyte as the excited state is more polar than the ground state and can, therefore, interact with solvent molecules via dipole-dipole interactions.  Changing solvent from a non-polar (i.e. hexane) to a polar (i.e. ethanol) will result in changes in the absorbance maximum.
  • For impurity quantification relative response factors need to be calculated, hence, pure standards must be available which is not always possible. 
 

Applications

UV-visible detectors are utilized in a myriad of different application areas including small organic molecule analysis, with biological macromolecules (peptides), and where any UV active molecule is present.

 
 

Diode Array Detector (DAD)

Diode array detectors (DAD) can be used for detection at single or multiple wavelengths.  Spectra can be dynamically obtained and stored for peak purity analysis, library searching, and extraction of signals (Figure 7).

Combined tungsten and deuterium lamps emit radiation from 190-850 nm.  Radiation is collimated through the flow cell then a mechanically controlled slit.  The radiation is dispersed at the holographic grating into individual wavelengths of light.  Each photodiode receives a different narrow wavelength band.  A complete spectrum is taken every ~12 ms and spectra signals are created and stored.

 

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Figure 7: Diode array detector (DAD).
 

The diode array detector allows two different wavelengths to be monitored concurrently which can be useful for many applications including peak purity analysis and peak suppression.  For peak purity analysis plotting the ratios of two absorbances gives a ratiogram (Figure 8) which can be used to determine if the peak is pure. If a rectangular ratiogram is obtained this indicates that the peak is pure.

However, a small caveat to this is that if an impurity elutes at the exact same time and has an identical shape or an identical UV spectrum, then peak purity cannot be determined in this way.  Many diode array detectors will also have in built functions for determining peak purity.  These settings must be manipulated for each specific sample and it is advisable to obtain training on the specific detector before using these settings.

It is also advisable that when peak purity is required to be determined conclusively other methods of analyte identification are used, i.e. infrared (IR), nuclear magnetic resonance (NMR), mass spectroscopy etc.  Another way to determine peak purity is to use an alternative separation process, e.g. reversed-phase HPLC followed by normal-phase HPLC.

 

Figure 8: Ratiogram used for determining peak purity.

 

There are several online libraries in which UV spectra of known compounds are stored.  Spectra taken of individual peaks during a HPLC-DAD experiment can be obtained and stored for comparison with these libraries.  The acquisition time of a diode array detector is very quick (less than half a second including data processing), therefore, spectra for numerous peaks within a run can be easily obtained. 

 
 

Bandwidth and Slit Width

Bandwidth is related to the number of diode responses which are averaged in order to obtain a signal at a particular wavelength.  A wide bandwidth results in a larger range of wavelengths being averaged when producing a spectral data point which results in a loss of spectral resolution, however, noise will be reduced and, therefore, sensitivity will increase (Figure 9).  Conversely a narrow bandwidth produces spectra with high spectral resolution and an increase in noise accompanied by a reduction in sensitivity.

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Figure 9: DAD bandwidth settings.
 
 

Some diode array detectors have a variable slit at the entrance to the spectrograph.  A narrow slit width provides improved spectral resolution for analytes which give UV spectra with enough fine detail to be useful for qualitative analysis (Figure 10).

Improved spectral resolution can be useful in library matching.  A wide slit width allows more of the light passing through the flow cell to reach the photodiodes, hence, the signal intensity and detector sensitivity increase.  Furthermore, baseline noise is also reduced.  However, with a wide slit width the wavelength of light falling on each diode becomes less specific as the light becomes more diffuse resulting in a loss of spectral resolution.

 

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Figure 10: DAD slit width settings.
 

The effect of modifying bandwidth and slit width is summarized below (Figure 11).

 
Figure 11: Summary of altering DAD bandwidth and slit width settings.
 
 

Response Time

Response time describes how fast the detector signal follows a sudden change of absorbance in the flow cell.  Decreasing the response time effectively allows the detector to take more measurements per unit time giving a better defined chromatogram (Figure 12).  Low response times are characterized by increases in peak height or area as well as a noisier baseline.  Increasing the response time results in averaging more data points and, therefore, reduces the noise by the square root of the number of data points. 

The drawback to increasing the response time is a slight loss in the peak height or area.  Response time again results in a compromise between how well the chromatographic signal and UV spectrum are defined (i.e. spectral resolution) and the sensitivity (S/N ratio). 

Generally 20-25 data points across a chromatographic peak are required for accurate quantification.

 

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Figure 12: Influence of response time on spectral quality.
 

Improvements have been made to UHPLC systems to increase the data acquisition rate of the detectors used.  An artefact of UHPLC separations is the narrow peak widths associated with highly efficient peaks.  In order to generate a truly representative peak to be used for a qualitative separation a minimum of 15 readings across the peak width are required.  When a chromatographic peak is going to be used for quantitative measurements a minimum of 20 and ideally 25 measurements across the peak width should be made.  The peak width is usually defined as the distance between the points where lines drawn tangentially to the sides of the chromatographic peak intersect the baseline.

In conventional HPLC separations (150 x 4.6mm, 5μm), where peak widths of 30 seconds are common, a data acquisition rate of 1 Hz (1 measurement per second) often suffice.  In UHPLC separations (100 x 2.1mm, <3μm core-shell or <2μm fully porous), when peak widths of 2 seconds are more common, a data acquisition rate of a 12 Hz or 0.08 seconds or 0.001 minute are required.   If running really high efficiency separations which are generating peak widths of 0.5 seconds (or even less in some cases) a data acquisition rate of 50 Hz or 0.02 seconds or 0.0003 minutes are necessary. 

Figure 13 demonstrates the effects of using different detector acquisition rates on analyte peak width and resolution.  The effects of ‘peak broadening’ due to low data acquisition rates are obvious and the phenomenon is often mistaken for physical peak dispersion!

 

Figure 13: Effect of data acquisition rate on chromatographic peak width and resolution.

 
 

Reference Wavelength

The reference wavelength compensates for fluctuations in lamp intensity as well as changes in the absorbance/refractive index of the background (i.e. mobile phase) during gradient elution.  During gradient elution the composition of the eluent will change and, hence, so will its refractive index.  This usually manifests itself as a gradual increase in response during the gradient time.  To compensate for the change in refractive index properties a reference wavelength should always be set otherwise drifting baselines will occur (Figure 14).

Noise will also be reduced as the reference wavelength is moved closer to the sample signal.  Without any reference measurement all noise and variability in lamp intensity is recorded within the signal.  When using a reference signal all lamp intensity and background (mobile phase) variability is subtracted out of the signal being measured.  The closer the reference wavelength is to the sample wavelength the more effectively these background deviations are catered for and the better the detector sensitivity.

However, the reference wavelength should not be selected too close to the analyte wavelength or the signal intensity may be seriously reduced.  Correct choice of a proper reference wavelength can reduce variability and drift in the chromatographic baseline resulting in better signal to noise performance.

 

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Figure 14: Chromatogram with reference wavelength set (top) and without reference wavelength set (bottom).
 
 

Sample and Reference Settings
A diode array detector can compute and store several signals simultaneously and also manipulate the signals together in order to yield a composite or deconvoluted chromatogram.  The following signals are usually collected using diode array detectors:

  • Sample wavelength – the center of a wavelength band with the width of the sample bandwidth
  • Reference wavelength – the center of a wavelength band with the width of the reference bandwidth
The signals comprise a series of data points over time with the average absorbance in the sample wavelength band minus the average absorbance of the reference wavelength band.  An empirical method is detailed below for setting typical sample and reference wavelengths.
 

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Figure 15: Setting sample and reference wavelengths on a diode array detector.
 
 

Peak Suppression

An interesting application of the diode array detector is that of peak suppression.  With peak suppression a reference wavelength is chosen so that the contribution from one analyte will be subtracted from another.  Interactive Figure 16 can be used to examine the suppression of caffeine and hydrochlorothiazide in a sample.  In this example to suppress caffeine the reference wavelength was set to 282 nm. 

At this wavelength caffeine shows the exact same absorbance as at 222 nm (the analytical wavelength for hydrochlorothiazide).  As hydrochlorothiazide does not absorb at all at 282 nm and the absorbance values are subtracted from each other any indication of the presence of caffeine is eliminated without affecting the analyte signal.

 

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Figure 16: Peak suppression using a diode array detector.
 
 

Diode Array Detector – Advantages, Disadvantages, and Applications

Advantages
  • Simultaneous multi-wavelength detection can be carried out which allows for applications such as peak purity analysis, spectral library searching, peak suppression, and the creation of extracted signals
  • Fast scan speeds
  • Interfering peaks can be eliminated using peak suppression
  • Peak purity can be estimated
Disadvantages
  • Susceptible to lamp fluctuations
  • Peak purity is only indicative and needs to be carefully set up and operated
  • Sensitivity is lower than single wavelength detectors
Applications
  • Peak purity
  • Library searching for structural elucidation
  • Broad bandwidth strategies can be employed that detect ‘any’ UV active components
 
 

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The interactive diagram in Figure 17 can be used to explain the various components of a fluorescence detector.  The most important parameters to be optimized in fluorescence detection are the excitation and emission wavelengths.  It is generally assumed that the ideal excitation wavelength can be taken from the excitation spectrum generated on a spectrofluorimeter.   sponsored by
 

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Figure 17: Fluorescence detector optical path.
 
 

Excitation and Emission Spectra

The excitation and emission spectra of quinidine are shown in Figure 18.  The excitation spectrum corresponds to the absorption of photons from the ground to the excited states of the molecule.  It will be very similar to the absorbance spectrum obtained with a diode array detector.  There will be some differences in the spectrum due to variations in the optical components of the detectors.

The emission spectra occur at higher wavelengths (lower energy) and correspond to the photon emission from the lowest level singlet state to the ground state of the molecule.  Spectra such as these can be collected for each component in the chromatogram to optimize the analysis for each individual component.
When determining the excitation and emission wavelengths for a particular analyte it is usual to first fix the excitation wavelength and then optimize the emission wavelength.

 

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Figure 18: Excitation and emission process and spectra.
 
 

Fluorescent molecules often have some of the following characteristics; conjugated aromatic systems, rigid structures (fused aromatic rings), and heteroatoms.  Organic molecules that tend to fluoresce are fluorescent proteins, dyes, and intrinsic fluorophores.  Inorganic molecules that are fluorescent including lanthanide containing compounds and quantum dots.  Some examples of fluorescent molecules are shown in Figure 19.

 
 

Acridine orange fluorescent dye

 

Fluorescent lanthanide containing complex

 

Aequorea victoria Green fluorescent protein

 

Quantum dot

Figure 19: Fluorescent molecules. 

 
 

Only a minority of molecules exhibit fluorescence which makes the fluorescence detector one of the most specific detectors available for HPLC analysis.  While only a minority of molecules naturally fluoresce it is possible to derivatize non-fluorescent molecules using fluorotags such as fluorescein isothiocyanate (FITC, Figure 20).

 

Figure 20: Protein fluorotagging using fluorescein isothiocyanate (FITC).

 
 

The sensitivity of a fluorescence detector can be up to 1000 times greater than a UV-visible detector with the minimum mass detected in the range of 10-100 pg (c.f. 0.5-1.0 ng for a UV-visible detector).  The linear range of this detector is system dependent and may be relatively small (parameters such as sample, solvent and accompanying components must be taken into account).  It should also be noted that unsuitable solvents and oxygen can quench fluorescence and should, therefore, be avoided.

Advantages, Disadvantages, and Applications

Advantages

  • High specificity (only a minority of molecules fluoresce)
  • Very sensitive (10-100 pg)
  • Very small volumes of sample are required

Disadvantages

  • Most molecules do not naturally fluoresce
  • Affected by temperature fluctuations (increased molecular collisions, decrease in potential energy)
  • Excitation and emission wavelengths may differ from instrument to instrument

Applications

  • Fluorescent labelled radionucleotides
  • Food additives (t-butylhydroquinone)
 
 

Analytical Chemists

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Initially both sample and reference cells are flushed with mobile phase (Figure 21).  The reference cell is then closed and solvent flows only through the sample cell.  The refractive index of the mobile phase in both cells is the same and the position of the zero glass can be adjusted so that the detector is in optical balance with an equal amount of light falling on each diode.

When the sample elutes from the column into the sample cell the refractive index of the cell contents changes.  The change in refractive index deflects the light beam as it passes through the flow cell resulting in an unequal amount of light reaching the photodiodes (Figure 22).

The change in current from the photodiodes is amplified and used to produce the calibrated detector signal.  The signal is expressed as nano Refractive Index Units (nRIU) and corresponds to the difference in refractive index of the sample (in the sample cell) and the mobile phase (in the reference cell).

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Figure 21: Refractive index detector.
 

The refractive index detector is a non-selective detector which is 1000 times less sensitive than a UV-visible detector; the minimum mass detected is 1-5 μg.  Refractive index detectors can work at very low flow rates with very low volume cells.  However, they exhibit high sensitivity to flow rate fluctuations and mobile phase composition as this will alter the refractive index properties of the bulk solution.
Some other factors that affect refractive index are:

Wavelength
The refractive index varies with changes in the wavelength of the incident light beam.

Density
As the density of the medium changes the refractive index also changes.  At a fixed wavelength of incident light the changes in refractive index are generally linear in relation to the changes in medium density.
The density of a medium will be affected by the following factors:

  • Composition
  • Temperature – the refractive index of a liquid changes by approximately 5 x 10-4 units per oC
  • Pressure

Refractive index detectors are not normally compatible with gradient elution; however, research efforts in the field of detector technology have demonstrated advances in the use of refractive index detectors that can be used for gradient analysis.1-3

 
 

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Figure 22: Refraction of light with a reference only and with a sample.

 

Advantages, Disadvantages, and Applications

Advantages

  • Good for the detection of non-ionic compounds, analytes that do not have a UV-visible chromophore and molecules that do not fluoresce
  • It is a universal detector for routine HPLC operation

Disadvantages

  • Not suitable for gradient analysis (eluent composition must remain constant throughout analysis)
  • Affected by changes in flow rate and temperature
  • Long equilibration times are required
  • Temperature dependent

Applications

  • Food analysis (carbohydrates)
  • Gel permeation chromatography (GPC)
 
 

Analytical Chemists

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The Evaporative Light Scattering detector has three primary regions; nebulization, desolvation, and detection.  Within the low temperature nebulization area the mobile phase leaving the HPLC column is nebulized using an inert pressurized gas, either air or nitrogen, to form an aerosol (Figure 23).

The desolvation region evaporates the mobile phase to produce dried solute particles.  Evaporation occurs as a function of time, temperature, and pressure of the carrier gas.  It is important that the eluent used is fairly easily evaporated, therefore, solvents with fairly low boiling points and viscosities are preferable.  If the analyte is less volatile than the mobile phase it will remain in the gas stream as a dry solute particle. 

The particles scatter the light and the amount of scatter is measured to produce a signal.  The intensity of the scattered light is measured using a photomultiplier tube or photodiode.  The size of the particle determines how the light is scattered, with the detector measuring the intensity of the scattered light at 60o relative to the excitation beam; this minimizes polarization effects and stray light.

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Figure 23: Evaporative light scattering detector.

 

The size of the particles determines how the light is scattered.  Larger particles scatter more light and, therefore, produce a larger signal, and hence, chromatographic peak.  The output has no direct relation to the analyte molecular weight; however, it does bear a relationship to the concentration of the material that is eluted from the column.

 
 

Types of Light Scattering

There are three possible types of light scattering Rayleigh, Mie, and refraction-reflection.
If a nebulizer produces droplets with an average diameter of D0 the diameter of the resulting dry analyte particle (D) is given by Equation 2.

 

Where:
D0 = average liquid droplet diameter
c = concentration of the analyte
ρ = density of the dry analyte

 
 

The response from an ELS detector can be the result of all the possible light scattering types.  The type of light scattering that occurs for a dry solute particle is dependent on the particle size.  The ratio of the particle diameter (D) to the incident light wavelength (λ or D/ λ) defines the type of light scattering which occurs.

 
   
 
  • Occurs for the smallest particles where D/λ < 0.1
  • Scattered light is proportional to D6, therefore, the signal is proportional to c2
 
  • Occurs for particles where 0.1 < D/λ < 1.0
  • Scattered light is proportional to D4, therefore, the signal is proportional to c4/3
 
  • Occurs for particles where D/λ > 1.0
  • Scattered light is proportional to D2, therefore, the signal is proportional to c2/3
 

Advantages, Disadvantages, and Application

Advantages

  • Analysis of non-volatile analytes
  • Not influenced by UV or refractive index properties of the analyte, or changing eluent compositions, therefore, it is gradient compatible
  • Detects species with no chromophores

Disadvantages

  • The mobile phase must be volatile and the sample must not be
  • The detector response is a complex function of the injected amount of analyte
  • Small volatile molecules are difficult to analyze
  • The signal is not linear with analyte concentration

Applications

  • Drug discovery – especially for small polar and charged species
  • Natural product development
 
 

Analytical Chemists

  • I feel empowered to fix things
  • I can troubleshoot effectively
  • I know where to go for help
  • I understand my analyses
  • I know where to get applications
  • I’m up to date
  • I’m more employable
  • My career is progressing
 

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The eluent is nebulized using a pressurized gas (e.g. nitrogen) to form an aerosol of ultra-fine droplets (Figure 24).  Large droplets are removed by an impactor as these would not dry properly resulting in increased noise.

A stream of positively charged gas collides with the analyte particles transferring the charge; the larger the particles the greater the charge.  The charged particles then enter the collector ad are measured by an electrometer.  The signal produced is directly proportional to the quantity of the analyte present.

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Figure 24: Charged aerosol detector (CAD).  (Image reproduced with permission from Thermo Fisher Scientific, Waltham, MA, USA).

 

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Advantages, Disadvantages, and Applications

Advantages

  • Compatible with gradient elution
  • Universal (for sufficiently non-volatile solutes)
  • Good sensitivity (ng quantities)
  • Wide dynamic range

Disadvantages

  • Analyte should be non-volatile
  • A volatile mobile phase is required

Applications

  • Lipid analysis
  • Formulation development
  • Stability analysis
 
 

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Within the electrochemical cell a potential is applied across the working and counter (also called the auxiliary) electrode.  Electrochemically active species will move from the bulk media to the electrode surface by diffusion where the electrochemical reaction takes place.  The electrode potential is required to facilitate the electrochemical reaction.  The current that results from the electrochemical reaction is amplified and plotted as a function of time to give a chromatographic peak.  It is important to obtain the optimum potential in order to maximize the signal output.  If the potential is too low no electrochemical reaction will occur, therefore, there will be no peaks in the chromatogram.  A potential that is too high can cause spurious oxidation reactions (i.e. mobile phase oxidation) which will produce more noise and interfering peaks.    sponsored by

Electrochemical detectors consist of three electrodes; a working, reference, and counter electrode (Figure 25).

The applied potential drives the electrochemical reaction at the surface of the working electrode.  Working electrodes are often made of carbon based materials; glassy carbon, pyrolytic carbon, and porous graphite.  Metals such as platinum, gold, silver, nickel mercury, gold-amalgam and a variety of alloys are also commonly used.  The choice of working electrode is dependent on the applied potential required, involvement of the electrode in the redox reaction, and the kinetics of the electron transfer reaction.  Compatibility of the mobile phase with the working electrode material should also be considered. 

The counter (or auxiliary) electrode balances the current that is produced from the working electrode and is often made of platinum.

The reference electrode is used as a reference to which the working electrode is set, and defines the zero point on the potential axis.  Historically this electrode was most commonly a standard hydrogen electrode (SHE).  Other materials that have been used as reference electrodes include Ag/AgCl, solid silver wire coated in silver chloride, mercury/mercurous chloride, calomel, and hydrogen/palladium.

 

Figure 25: Three electrode electrochemical detector.

 

In UV-visible detection the Beer-Lambert Law describes the relationship between absorbance and analyte concentration.  In electrochemical detection the relationship between analyte concentration and peak height is given by the Cottrell Equation (Equation 3) which can be simplified to Equation 4.

 
 

Where:
ilim = current (i.e. peak height)
b = spacer thickness
C = concentration
F = Faraday constant, 9.649 x 104 Cmol-1
A = working electrode area

 
 

There are several types of electrochemical detectors which can be used for a variety of different applications.

Conductivity detection – this is the measurement of the electrical current carried by dissolved ions in an electric field.  It is commonly used for the detection of ionic species (organic/inorganic ions, and anions and cations of strong acids and bases).

DC amperometric detection – this involves the measurement of current resulting from the oxidation or reduction (electrolysis) of analytes at an electrode surface.  The major application of this type of detector is the detection of molecules containing phenol or catechol functional groups (i.e. catecholamines).

Integrated and pulsed amperometric detection – molecules are oxidized or reduced at an electrode surface.  The current is measured by integration during a repeated potential vs. time waveform.  This detector type is excellent for the analysis of carbohydrates.
For an electrochemical reaction to occur the analyte must be electrochemically active, some electrochemically functional groups are shown in Figure 26.  Furthermore, for electrochemical detection to occur the mobile phase must contain an electrolyte to permit the flow of current.

 

Figure 26: Electrochemically active functional groups.

 
 

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  1. McBrady, A. D.; Synovec, R. E.  J. Chromatogr. A 2006, 1105, 2.
  2. Lapsley, M. I.; Chiang, I. –K.; Zheng, Y. B.; Ding, X.; Mao, X.; Huang, T. J. Lab Chip 2011, 11, 1795.
  3. Wade, J. H.; Bailey, R. Anal. Chem. 2014, 86, 913.
 
 

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The selection of a detector for HPLC is based on the chemical nature of the analytes and any potential interferents that may be present, the limit of detection required, and often the availability and cost of the detector. This month’s Essential Guide will focus on the operating principles of the most common HPLC detectors. The parameters that should be optimized for each detector type will be examined to give the optimum performance from the chosen detector. The advantages, disadvantages, and application areas for each detector type will also be discussed. This in depth review of HPLC detectors will result in a better understanding of the operation, optimization, and detector choice for every application.

Scott Fletcher
Technical Business Development Manager
Crawford Scientific

Bob Boughtflower
Section Head, Analytical Technologies
GSK

Key Learning Objectives:

  • Understand the operating principles and limitations of common HPLC detectors
  • How to optimize detector settings to maximize analytical response
  • Identify the correct detector for a specific application
  • Understand the importance of using the correct detector settings for your analysis